Anthrax (Bacillus anthracis) is primarily a disease of domesticated and wild animals, particularly herbivorous animals, such as cattle, sheep, horses, mules, and goats. Although natural anthrax infection in humans is rare (risk of infection through contact with diseased animals is about 1/100,000), it poses a very real threat from bioterrorism. The bacteria form hardy spores which are heat resistant and can survive for decades under crude conditions. Although cutaneous anthrax is more readily treatable, inhalation anthrax typically results in an abrupt catastrophic illness having a mortality rate of greater than 80% in 2-4 days. The ease by which the disease can be spread due to the stability of the spores was made evident from the five deaths that occurred in the U.S. in 2002. If anthrax spores were spread through an act of terrorism, the event would likely be undiscoverable until large numbers of people sought treatment or died. Current therapies, including antibiotics and vaccines, cannot help most of these victims.
The current model of anthrax infection teaches that after gemination of spores, the actively growing bacterial cells produce a capsule containing poly-D-glutamate polypeptide, which protects the bacteria against the bactericidal components of serum and phagocytes and phagocytic engulfment. The capsule is more important during the establishment of the infection than in the terminal phases of the disease, which are mediated by the anthrax toxin. The toxin, which is responsible for the disease etiology, is composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). The EF is a calcium-calmodulin-dependent adenylate cyclase believed to cause the edema associated with anthrax infection and to prevent immune cells from ingesting and degrading the bacteria. The LF is a cell-type specific metalloprotease that cleaves mitogen-activated protein kinase-kinases and several peptide hormones. It causes macrophage cell death and release of toxic substances (e.g., those associated with septic shock such as TNF-α and IL-1). LF is the major virulence factor associated with anthrax toxicity and is responsible for systemic shock and death.
None of the toxin components are pathogenic alone, and EF and LF require PA to exert their toxic effects inside a host cell. During infection, an 83 kDa protective antigen PA83 protein secreted from rapidly growing B. anthracis binds to the host's cell surface via the anthrax toxin receptor (ATR) (Bradley et al, 2001, Nature 414:225-229). Cleavage of the PA83 by a membrane-bound furin and/or a furin-like protease releases an amino-terminal 20 kDa PA fragment, resulting in receptor-bound 63 kDa PA. Oligomerization of PA63 into a heptameric ring forms a high-affinity binding site recognized by the amino termini of both LF and EF. Endocytosis of the receptor-toxin complex into acidic endosomes elicits a conformational change in PA63 whereby LF or EF is released into the endosome. Lysosomal acidification and subsequent receptor release facilitates irreversible membrane insertion of the oligomeric PA63 pore. The pore formed by PA63 permits transport of LF and EF into the cytoplasm where they can elicit their respective toxicities.
Several modes of interrupting PA function have been explored in order to develop treatments against anthrax. Soluble ATR (sATR) introduced into media containing ATR-bearing cells (e.g., macrophages), causes PA to bind to the sATR instead of the receptor on the cell surface (Bradley et al., 2003, Biochem. Pharmacol. 65: 309-314) suggesting that ATR may be useful in the design of anthrax treatments. It has also been shown that mutant forms of PA called dominant negative inhibitors (DNIs) permit formation of the heptameric complex when mixed with native PA, which was unable to inject the EF and LF into the cell in both cell culture and rodent models (Sellman et al., 2001, Science 292: 695-697). Monoclonal antibodies generated against the PA may lessen the symptoms of infection as well as provide prophylaxis. An antibody may block the binding of PA to its receptor on the surface of the cell and/or it may block the EF or LF from binding to the PA. Either of these methods could prevent toxin from entering the cells and causing damage. While antibiotics alone may control bacterial expansion, they do nothing to neutralize the effects of the toxin. In addition, it may be possible to engineer resistance into the bacteria thereby rendering the antibiotic useless.
Anthrax vaccine (AVA), which contains PA as the primary immunogenic component, may confer protection against the disease. However, there are several drawbacks to using AVAs. The immunization schedule (e.g., six initial doses followed by yearly boosters) does not generate strong immunological memory. Also, a lack of standardization of the level of antigen results in a high degree of variability in efficacy on a lot-lot basis. Furthermore, since the vaccine is a cell-free culture media filtrate, which contains several cellular components, it may contribute to a high incidence of local and systemic reactions. In a prophylactic use of polyclonal antibodies generated in response to the vaccine, antibodies to PA have been shown to prevent disease following exposure to anthrax. Development of human scFv screened from a naïve single-chain Fv phagemid library for antibodies that bind PA have been described (Cirino et al., 1999, Infection and Immunity 67: 2957-2963). These PA binding agents were selected against purified PA83 based on their ability to inhibit receptor-mediated binding of PA to cells. However, since these immunological agents are not complete antibodies, their use in treatments is limited by a decreased half-life and lower avidity, which may require much higher dosages, particularly in prophylactic treatments.
Monoclonal antibodies have advantages over antibiotics, but also which can be used to augment antibody efficacy. For treatment of active pulmonary disease, antibiotics will not neutralize the preformed and released toxin that is causing the pathology, whereas antibodies can neutralize additional toxin before it can contribute further to the inflammatory cascade. For prophylactic treatment, the duration of required antibiotic prophylaxis is 60 days, which can be difficult to follow. Furthermore, this time span may be covered by a single injection of antibody. Moreover, in true exposures, antibiotics can interfere with the development of a protective immune response so that there is no protection afforded after dosing is terminated.
Accordingly, there is a need for improved therapies for treating anthrax infection, particularly antibodies against B. anthracis protective antigen, which will be well tolerated by the immune system and capable of prophylactic, post-exposure prophylactic, and therapeutic uses.